犬弓首蛔虫TcCPG1的表达及其与几丁质的结合特性

为探讨犬弓首蛔虫（Toxocara canis，T.canis）软骨素蛋白多糖TcCPG1的表达特征及其与几丁质的结合方式，为犬弓首蛔虫软骨素蛋白多糖的功能研究提供依据，笔者克隆T.canis软骨素蛋白多糖1基因（Tc-cpg-1）并原核表达重组蛋白TcCPG1，采用镍柱亲和层析技术对重组蛋白进行纯化，并与几丁质进行结合试验；采用实时荧光定量PCR（quantitative real-time polymerase chain reaction，qRT-PCR）进行Tc-cpg-1基因的转录水平的性别和组织差异表达分析。结果显示：Tc-cpg-1基因编码区（coding sequence，CDS）的长度为1 251 bp，共编码417个氨基酸；SDS-PAGE电泳结果显示重组蛋白TcCPG1以包涵体形式存在，相对分子质量大小约为70 ku；对表达条件进行优化后，以0.8 mmol·L^-1 IPTG于16℃诱导14 h时可获得大量目的蛋白；利用镍柱亲和层析纯化可以获得具有较高纯度的目的蛋白，与几丁质进行结合试验，验证了重组蛋白TcCPG1能够与几丁质结合；通过qRT-PCR对Tc-cpg-1基因转录水平的组织差异性进行分析，发现Tc-cpg-1在雌虫中高量表达，尤其在雌虫的生殖组织中表达量最高，表明Tc-cpg-1可能参与了T.canis雌虫的生殖过程，推测TcCPG1可能通过与卵壳中几丁质的相互作用从而影响T.canis的胚胎和生殖发育过程。     

Preliminary Studies on CPG/Hinf Ⅰ RFLP Groups of Citrus tristeza virus Infected Sweet Oranges in China

Citrus tristeza virus （CTV） causes economically important losses to the citrus industry worldwide. Mild strain cross protection （MSCP） against tristeza has hardly been practised due to mixed infection of different CTV-strains and little background of its molecular biology in China. For better cognition on CTV, 192 sweet orange samples collected from eight provinces （Chongqing, Sichuan, Fujian, Hunan, Guangxi, Yunnan, Guangdong and Jiangxi） were tested by direct tissue blot immuno-assay （DTBIA）, and 158 of them were tested positively, which therefore were subjected to coat protein gene （CPG）/Hinf Ⅰ restriction fragment length polymorphism （RFLP） analysis. Sample bulks were compared between Chongqing and Fujian by some statistical data, including ratios of single infection and mixed infection to local samples, proportions of CTV isolates with single RFLP groups, and rates of each RFLP group. The simplified analysis of samples from the other six provinces were then conducted. This study suggests that CTV isolates with CPG/Hinf Ⅰ RFLP groups Ⅲ and Ⅰ are the main epidemic ones in China, and mixed infection of CTV in fields are popular. Based on observation of severity of stem-pitting symptom in field trees, CTV isolates with CPG/Hinf Ⅰ RFLP groups Ⅲ and Ⅰ caused severe stem-pittings in sweet oranges in China.